Evaluation of the Inhibitory Effects of Pyridylpyrazole Derivatives on LPS-Induced PGE2 Productions and Nitric Oxide in Murine RAW 264.7 Macrophages.

A series of thirteen triarylpyrazole analogs were investigated as inhibitors of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 macrophages. The target compounds 1a-m have first been assessed for cytotoxicity against RAW 264.7 macrophages to determine their non-cytotoxic concentration(s) for anti-inflammatory testing to make sure that the inhibition of PGE2 and NO production would not be caused by cytotoxicity. It was found that compounds 1f and 1m were the most potent PGE2 inhibitors with IC50 values of 7.1 and 1.1 µM, respectively. In addition, these compounds also showed inhibitory effects of 11.6% and 37.19% on LPS-induced NO production, respectively. The western blots analysis of COX-2 and iNOS showed that the PGE2 and NO inhibitory effect of compound 1m are attributed to inhibition of COX-2 and iNOS protein expression through inactivation of p38.

Synthesis and evaluation of multivalent nitroimidazole-based near-infrared fluorescent agents for neuroblastoma and colon cancer imaging.

Hypoxia is one of key characteristics of microenvironments of solid tumors, and evaluation of hypoxia status in solid tumors is important to determine cancer stage and appropriate treatment. In the present study, novel, multivalent, near-infrared (NIR) fluorescent imaging agents were developed to measure tumor hypoxia. These agents were synthesized using an amino acid as a backbone to connect mono-, bis-, or tris-2-nitroimidazole as a hypoxia-sensitive moiety to enhance uptake by the tumor and to attach sulfo-Cyanine 5.5 as an NIR fluorophore to visualize tumor accumulation. Studies of physical characteristics demonstrated that the novel NIR imaging agents showed suitable optical properties for in vitro and in vivo imaging and were stable in serum. In vitro cellular uptake studies in SK-N-BE(2) and SW620 cell lines demonstrated that NIR imaging agents bearing 2-nitroimidazole structures showed significantly higher tumor uptake in hypoxic cells than in normoxic cells. Moreover, in vivo optical imaging studies using SK-N-BE(2) and SW620 xenografted mice demonstrated that novel, multivalent, 2-nitroimadazole NIR imaging agents with two or three 2-nitroimidazole moieties showed higher uptake in tumor than the control agents with only one 2-nitroimidazole. These observations suggest that novel, multivalent, NIR agents could serve as potential optical imaging agents for evaluating tumor hypoxia.

Novel multifunctional 18F-labelled PET tracer with prostate-specific membrane antigen-targeting and hypoxia-sensitive moieties.

Prostate cancer is one of the most frequently found cancers in men worldwide. Prostate-specific membrane antigen (PSMA) is typically highly expressed in prostate cancer, and the Glu-Urea-Lys (GUL) structure has recently received considerable attention as a key unit of PSMA-targeting agents. Additionally, one of the common characteristics of many solid tumors, such as prostate cancer, is hypoxia. In this study, novel multifunctional PSMA inhibitors containing a PSMA-targeting moiety either with or without a hypoxia-sensitive moiety (18F-PEG3-ADIBOT-2NI-GUL and 18F-PEG3-ADIBOT-GUL, respectively; ADIBOT: azadibenzocyclooctatriazole, 2NI: 2-nitroimidazole) were designed and synthesized, and their feasibility as PET tracers for prostate cancer imaging studies was examined. The compounds labelled with 18F via the copper-free click reaction were stable in human serum and showed nanomolar binding affinities in in vitro PSMA binding assays. Micro-PET and biodistribution studies indicate that both 18F-labelled inhibitors successfully accumulated in prostate cancer regions, and 18F-PEG3-ADIBOT-2NI-GUL showed a 2-fold higher tumor-to-total non-target organ ratio than that of 18F-PEG3-ADIBOT-GUL, suggesting that the synergistic effects of the PSMA-targeting GUL moiety and the hypoxia-sensitive 2-nitroimidazole moiety can increase tumor uptake of the novel PET tracers in prostate cancer. These findings suggest that this novel multifunctional PET tracer with an 18F-labelled PSMA inhibitor and a 2-nitroimidazole moiety is a potent candidate to provide better diagnosis of prostate cancer via PET imaging studies.

Synthesis and evaluation of novel potent TSPO PET ligands with 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide.

Translocator protein (TSPO) expression is closely related with neuroinflammation and neuronal damage which might cause several central nervous system diseases. Herein, a series of TSPO ligands (11a-c and 13a-d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714. Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity for in vivo brain studies. After radiolabeling with fluorine-18, [18F]11a was used for a dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation model; the inflammatory lesion was clearly visualized with a superior target-to-background ratio compared to [18F]DPA-714. An immunohistochemical examination of the dissected brains confirmed that the uptake location of [18F]11a in the PET study was consistent with a positively activated microglia region. This study proved that [18F]11a could be employed as a potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis of other diseases, such as cancers related with TSPO expression.

Synthesis and Evaluation of Multifunctional Fluorescent Inhibitors with Synergistic Interaction of Prostate-Specific Membrane Antigen and Hypoxia for Prostate Cancer.

Prostate cancer is one of the most common cancers in the world. It is widely known that prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and hypoxia is a common characteristic of many solid tumors, including prostate cancer. In this study, we designed multifunctional fluorescent inhibitors to target PSMA and tumor hypoxia in order to increase the tumor uptake of inhibitors. Novel PSMA inhibitors were prepared using lysine as the backbone to connect three different functional groups: the glutamate-urea-lysine (GUL) structure for inhibiting PSMA, 2-nitroimidazole for the hypoxia-sensitive moiety, and a near-infrared fluorophore (sulfo-Cyanine 5.5). According to the in vitro PSMA binding assay, novel fluorescent inhibitors were demonstrated to have nanomolar binding affinities. Multifunctional inhibitor 2 with one 2-nitroimidazole had a similar inhibitory activity to inhibitor 1 that did not contain the hypoxia targeting moiety, but multifunctional inhibitor 3 with two 2-nitroimidazoles showed lower inhibitory activity than inhibitor 1 due to the bulky structure of the hypoxia-sensitive group. However, in vivo optical imaging and ex vivo biodistribution studies indicated that both multifunctional inhibitors 2 and 3 had higher accumulation in tumors than inhibitor 1 due to a synergistic combination of PSMA and hypoxia targeting moieties. These observations suggest that this novel multifunctional strategy might be a promising approach to improve the diagnosis and therapy of prostate cancer.

Novel potential pyrazolopyrimidine based translocator protein ligands for the evaluation of neuroinflammation with PET.

Translocator protein (TSPO) is an interesting biological target because TSPO overexpression is associated with microglial activation caused by neuronal damage or neuroinflammation, and these activated microglia are involved in several central nervous system diseases. Herein, novel fluorinated ligands (14a-c and 16a-c) based on a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide scaffold were synthesized, and in vitro characterization of each of the novel ligands was performed to elucidate structure activity relationships. All of the newly synthesized ligands displayed nano-molar affinity for TSPO. Particularly, an in vitro affinity study suggests that 2-(5,7-diethyl-2-(4-(3-fluoro-2-methylpropoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (14a), which exhibited high nano-molar affinity for TSPO and proper lipophilicity, was suitable for in vivo brain studies. Thus, radiosynthesis from tosylate precursor 13a using fluorine-18 was performed, and [18F]14a was obtained in a 31% radiochemical yield (decay-corrected). Dynamic positron emission tomography (PET) imaging studies were performed in a lipopolysaccharide (LPS)-induced neuroinflammation rat model using [18F]14a to identify the location of inflammation in the brain with a high target-to-background signal ratio. In addition, we validated that the locations of inflammatory lesions found by PET imaging were consistent with the locations observed by histological examination of dissected brains using antibodies. These results suggest that [18F]14a is a novel promising PET imaging agent for diagnosing neuroinflammation, and it may also prove to be applicable for diagnosing other diseases, including cancers associated with altered TSPO expression, using PET techniques.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates.

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation.

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12-14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. Ki values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.

Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques.

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.

Eliminating antibody polyreactivity through addition of N-linked glycosylation.

Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.

Binding mode characterization of NBD series CD4-mimetic HIV-1 entry inhibitors by X-ray structure and resistance study.

We previously identified two small-molecule CD4 mimetics--NBD-556 and NBD-557--and synthesized a series of NBD compounds that resulted in improved neutralization activity in a single-cycle HIV-1 infectivity assay. For the current investigation, we selected several of the most active compounds and assessed their antiviral activity on a panel of 53 reference HIV-1 Env pseudoviruses representing diverse clades of clinical isolates. The selected compounds inhibited tested clades with low-micromolar potencies. Mechanism studies indicated that they act as CD4 agonists, a potentially unfavorable therapeutic trait, in that they can bind to the gp120 envelope glycoprotein and initiate a similar physiological response as CD4. However, one of the compounds, NBD-09027, exhibited reduced agonist properties, in both functional and biophysical studies. To understand the binding mode of these inhibitors, we first generated HIV-1-resistant mutants, assessed their behavior with NBD compounds, and determined the X-ray structures of two inhibitors, NBD-09027 and NBD-10007, in complex with the HIV-1 gp120 core at ∼2-Šresolution. Both studies confirmed that the NBD compounds bind similarly to NBD-556 and NBD-557 by inserting their hydrophobic groups into the Phe43 cavity of gp120. The basic nitrogen of the piperidine ring is located in close proximity to D368 of gp120 but it does not form any H-bond or salt bridge, a likely explanation for their nonoptimal antagonist properties. The results reveal the structural and biological character of the NBD series of CD4 mimetics and identify ways to reduce their agonist properties and convert them to antagonists.

MicroRNAs overexpressed in ovarian ALDH1-positive cells are associated with chemoresistance.

BACKGROUND: Ovarian carcinoma is the leading cause of cancer death worldwide among gynecological malignancies, and the majority of cases are related with recurrence and chemoresistance. Cancer stem cells (CSCs) are believed to be one of the causes of recurrent or chemoresistant ovarian cancer, and microRNAs are regulatory molecules newly implicated to control a variety of cellular processes, including CSCs. Therefore, we identified ovarian CSC-specific microRNAs and investigated their clinicopathological implication in ovarian carcinomas. METHODS: We isolated ALDH1 (+) cell population using the Aldefluor assay, and examined the differential expression pattern of miRNAs between ALDH1 (+) and ALDH1 (-) cells using a high-throughput microRNA microarray. We further investigated the expression patterns of differentially expressed miRNAs in human ovarian cancer samples using the real-time reverse transcription-polymerase chain reaction and analyzed their clinical impact in patients with ovarian cancer. RESULTS: We found that high ALDH1 expression was associated with chemoresistance in in vitro and ex vivo samples (p = 0.024). We identified six miRNAs, including miR-23b, miR-27a, miR-27b, miR-346, miR-424, and miR-503, overexpressed in ALDH1 (+) cells, and they were significantly upregulated in chemoresistant ovarian cancer cells (1.4 ~ 3.5-fold) and tumor samples (2.8 ~ 5.5-fold) compared with chemosensitive group. Upregulation of ALDH1 (p = 0.019) and miR-503 (p = 0.033) correlated with high clinical stage, and upregulation of miR-27a was related with distant metastasis (p = 0.046) in patients with ovarian cancer. CONCLUSION: Our findings indicate that ALDH1 is a useful marker for enriching ovarian CSCs, and high expression of ALDH1 and its related miRNAs, particularly miR-23b, miR-27b, miR-424, and miR-503, are significantly implicated in chemoresistance and tumor progression in ovarian cancer.

Effects of corticosteroid on the expressions of neuropeptide and cytokine mRNA and on tenocyte viability in lateral epicondylitis.

BACKGROUND: The purpose of this study was to determine the reaction mechanism of corticosteroid by analyzing the expression patterns of neuropeptides (substance P (SP), calcitonin gene related peptide (CGRP)) and of cytokines (interleukin (IL)-1α, tumor growth factor (TGF)-ß) after corticosteroid treatment in lateral epicondylitis. In addition, we also investigated whether corticosteroid influenced tenocyte viability. METHODS: The corticosteroid triamcinolone acetonide (TAA) was applied to cultured tenocytes of lateral epicondylitis, and the changes in the mRNA expressions of neuropeptides and cytokines and tenocyte viabilities were analyzed at seven time points. Quantitative real-time polymerase chain reaction and an MTT assay were used. RESULTS: The expression of SP mRNA was maximally inhibited by TAA at 24 hours but recovered at 72 hours, and the expressions of CGRP mRNA and IL-1α mRNA were inhibited at 24 and 3 hours, respectively. The expression of TGF-ß mRNA was not significant. Tenocyte viability was significantly reduced by TAA at 24 hours. CONCLUSIONS: We postulate that the reaction mechanism predominantly responsible for symptomatic relief after a corticosteroid injection involves the inhibitions of neuropeptides and cytokines, such as, CGRP and IL-1α. However the tenocyte viability was compromised by a corticosteroid.

Design, synthesis, and antiviral activity of entry inhibitors that target the CD4-binding site of HIV-1.

The CD4 binding site on HIV-1 gp120 has been validated as a drug target to prevent HIV-1 entry to cells. Previously, we identified two small molecule inhibitors consisting of a 2,2,6,6-tetramethylpiperidine ring linked by an oxalamide to a p-halide-substituted phenyl group, which target this site, specifically, a cavity termed "Phe43 cavity". Here we use synthetic chemistry, functional assessment, and structure-based analysis to explore variants of each region of these inhibitors for improved antiviral properties. Alterations of the phenyl group and of the oxalamide linker indicated that these regions were close to optimal in the original lead compounds. Design of a series of compounds, where the tetramethylpiperidine ring was replaced with new scaffolds, led to improved antiviral activity. These new scaffolds provide insight into the surface chemistry at the entrance of the cavity and offer additional opportunities by which to optimize further these potential-next-generation therapeutics and microbicides against HIV-1.

Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells.

MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

Interleukin-1ß induces angiogenesis and innervation in human intervertebral disc degeneration.

Degenerative disorders of the intervertebral discs (IVDs) are generally characterized by enhanced matrix degradation, angiogenesis, innervation, and increased expression of catabolic cytokines. In this study, we investigated the effects of inflammatory cytokines, IL-1ß, and TNF-α, on the expression of an angiogenic factor, vascular endothelial growth factor (VEGF), and neurotrophic factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), in human IVD degeneration. IL-1ß and TNF-α stimulated the gene expression of VEGF, NGF, and BDNF in nucleus pulposus (NP) cells isolated from patient tissues. Immunohistochemical results demonstrated a positive correlation between IL-1ß and VEGF/NGF/BDNF expression in human IVD tissues. RNA expression analysis of patient tissues also identified positive correlations between VEGF and platelet endothelial cell adhesion molecule-1 (PECAM-1) and between NGF/BDNF and protein gene product 9.5 (PGP9.5). Our findings suggest that IL-1ß is generated during IVD degeneration, which stimulates the expression of VEGF, NGF, and BDNF, resulting in angiogenesis and innervation.

FGF2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of JNK signaling.

FGF2 has been shown to enhance proliferation and maintain differentiation potential in hMSCs during in vitro propagation. In this study, we investigated the role of mitogen-activated protein kinase in the functions of FGF2 in hMSCs. We demonstrated that FGF2 induces the transient activation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 protein kinase. SP600125 and a dominant negative JNK1 significantly reduced the FGF2-enhanced proliferation of hMSCs. Treatment with SP600125 also diminished the activity of FGF2 in the maintenance of adipogenic and osteogenic differentiation potential. These results suggest that JNK signaling is involved in the FGF2-induced stimulation of the proliferation and the maintenance of differentiation potential in hMSCs.

Potential side effects of dendritic cells pulsed with allogenic melanoma cell lysate in mice.

An attempt has been made to investigate the toxicity of cancer immunotherapy based on the dendritic cells pulsed with lysate of allogenic melanoma cell, DM401. Dendritic cells pulsed with lysate of clone M3 were subcutaneously administered once a week eight times to C57BL/6 mice at 0, 2.5, 5, and 10 x 10(7) cells/kg. No changes attributable to the administration were observed in clinical signs and food and water consumption. The administration induced slight increases in body weights, white blood cells, total protein, total cholesterol, triglyceride, phospholipids, and absolute spleen weights, but a slight decrease in albumin/globulin ratio. Microscopic examinations revealed the infiltration of inflammatory cells in the lung, mainly in the pulmonary arteriole, in which the tunica media thickened, and in the pulmonary alveoli and alveolar space. Thickened tunica media of pulmonary arteriole was observed in both males and females at all selected doses. In addition, the subcutis at the test substance-application site showed inflammation and fibrosis. In conclusion, lung is a target organ of DM401, and most of the changes including the findings in lung are considered as the immunomodulatory functions of dendritic cells.

Crystal structures of the Rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly.

To understand the role of the pro-peptide in proteasome assembly, we have determined structures of the Rhodococcus proteasome and a mutant form that prevents the autocatalytic removal of its pro-peptides. The structures reveal that the pro-peptide acts as an assembly-promoting factor by linking its own beta-subunit to two adjacent alpha-subunits, thereby providing a molecular explanation for the observed kinetics of proteasome assembly. The Rhodococcus proteasome has been found to have a substantially smaller contact region between alpha-subunits compared to those regions in the proteasomes of Thermoplasma, yeast, and mammalian cells, suggesting that a smaller contact area between alpha-subunits is likely the structural basis for the Rhodococcus alpha-subunits not assembling into alpha-rings when expressed alone. Analysis of all available beta-subunit structures shows that the contact area between beta-subunits within a beta-ring is not sufficient for beta-ring self-assembly without the additional contact provided by the alpha-ring. This appears to be a fail-safe mechanism ensuring that the active sites on the beta-subunits are activated only after proteasome assembly is complete.